Cytokine is a general term for cell-secreted small proteins with biological activities. In many circumstances, the interactions among various immune cells are mediated by cytokines.
Among these cytokines, nerve growth factor (NGF) is a complex consisting of three different subunits, α, β, and γ, with a molecular weight of about 140 Kd. Researches show that the β subunit of NGF is a homodimer, with a single chain of 118 amino acid residues (Greene, L. A. and E. M. Shooter, The nerve growth factor: biochemistry, synthesis, and mechanism of action. Annu Rev Neurosci, 1980. 3: p. 353-402). The β subunit, the functional subunit of NGF, is in close association with many aspects, such as neuron survival, migration, growth, and differentiation, as well as establishing functional connections with other cells, and the like; has the dual functions of neurotrophy and neurite promoting; and is one of the most important biological active substances in nervous system. In addition, it also plays a vital role in preventing denervation muscle atrophy (Sofroniew, M. V., C. L. Howe, and W. C. Mobley, Nerve growth factor signaling, neuroprotection, and neural repair. Annu Rev Neurosci, 2001. 24: p. 1217-81). Clinical applications confirmed that local administration can enable nerves to obtain nutrition directly, which is beneficial for the axoplasmic flow of nerves, promoting nerve regeneration. Currently, the major administration methods for treating peripheral nerve injuries using neurotrophic factors are as follows: (1) direct administration at a local injury; (2) slow releasing administration by micro-osmotic pump at a local injury; and (3) transplanting cells expressing neurotrophic factors to a local injury via a transgenic technique. Application of exogenous β-NGF in vivo by means of direct administration is affected by many factors. The sustaining of its activity and its effect are associated with administration routes, modes and dosage. Single dose administration can barely ensure the continuous action of medicines, whereas successive administration requires a large amount of medicines, its treatment cost is very large, and its safety becomes a concern due to the large quantity of NGF. Micro-osmotic pump has problems of a rejection reaction and a poor absorption in vivo. The vector selection and safety issues for gene therapy are difficult to resolve. Therefore, all the administration methods mentioned above are difficult to apply NGF clinically.
Platelet-derived growth factor (PDGF-BB) is a peptide growth factor produced by various cell (e.g. platelet, mononuclear macrophage, vascular endothelial cell, vascular smooth muscle cell, placental and embryonic cells, mesangial cell and so on), and its biological properties are mainly showed as: 1) cell division promoting effects, capable of stimulating meiosis of various cells (e.g. vascular smooth muscle cells, fibroblasts, endothelial cells and glial cells), and regulating the renewal of extracellular matrix by stimulating collagen synthesis and activating the collagenase; 2) chemotaxis towards fibroblasts, smooth muscle cells and neutrophilic granulocytes; and 3) vasoconstriction effects. PDGF-BB plays an important role in cell culture, the treatment of skin ulcer, and the preparation of cosmetic additives. Currently, application in vivo of exogenous β-NGF by means of direct administration is affected by many factors. The sustaining of its activity and its effect are associated with routes, modes and dosage of administration. Single dose administration can barely ensure the continuous action of medicines whereas successive administration requires a large amount of medicines, its treatment cost is very large, and its safety becomes a concern due to the large quantity of PDGF-BB used.
Basic fibroblast growth factor (bFGF) is a multi-function peptide growth factor with the functions of promoting proliferation, migration and differentiation of various cells. It not only plays an important role in the early development of embryo, but also can promote the restoration of adult injury, and thereby is used for treating body injuries. Now, there are genetic engineered bFGFs on the market, which have been approved by the Department of Health, showing therapeutic values. However, current bFGFs used clinically all have the following defects: 1) easy to diffuse and invade into other tissues, causing injured sites not to be restored as desired, and other normal tissues to face potential safety issue, with increases in dosage and cost; and 2) a short half-life, easy to lose activity in vivo, causing decreased treatment effects.
Bone defects caused by injury, infection, tumor, and dysplasia are the problems encountered daily in clinical orthopaedics. Bone morphogenetic protein (BMP) belongs to transforming growth factor β superfamily. It can induce new bone formation in non-bone tissues (e.g. muscle), and thus is very important in bone growth and treatments of bone defects. BMP family includes at least 20 members, wherein human bone morphogenetic protein (BMP2) has a strong osteoinductive activity. Human BMP2 is a glycosylated protein of 396 amino acid residues in full length, including a signal peptide comprised of 19 amino acid residues, a pro-region consisting of 263 amino acid residues, and a mature peptide consisting of 114 amino acid residues. The mature peptide comprises seven cysteine residues and one N-glycosylation site, and its functional form is a homodimer formed through a pair of disulfide bond, the remaining six cysteine residues in each monomer forming three intrachain disulfide bonds (Wozney, J. M. et al. Novel regulators of bone formation: molecular clones and activities. Science 242, 1528-34, 1988). However, the content of natural BMP2 is very low, and isolation and purification of BMP2 from human and animal bones are of great limitations. At present, studies are focused on preparing recombinant BMP2 by means of genetic engineering, in order to meet the demands of clinical and fundamental researches. In addition, other factors functionally similar to BMP2, which can induce tissue regeneration and injury restoration, further include: BMP3, PDGF, FGF, EGF, TGF, VEGF, NGF, NT3/4 and the like.
Collagen, one of the major components of extracellular matrix of neurons, constitutes the scaffold for neuron growth together with other extracellular matrix. Combined with PDGF, Collagen is often used for treating skin injuries, such as ulcer. Additionally, collagen is a commonly used bone restoration material. It is a major organic component of bones, wherein type I collagen and its crosslinked fiber structure are the most abundant proteins within the extracellular matrix. Collagen structures can induce mineral deposition. There are mineral deposition sites on its surface, which can effectively induce and control the process of mineralization, promote bone formation, and induce it into implants. Via the molecular structures of collagen molecules, conventional collagen carriers prevent BMP2 component from diffusing and maintain the BMP2 concentration around host target cells. As such, on one hand, it is required that their pore sizes should not be too large. However, too small pore size is not good for the growth of osteoblasts, which requires the control of a range of pore sizes, introducing some difficulties in technology. On the other hand, the use of current biological materials as BMP2 carriers causes large dose of BMP2, usually up to a level of milligrams (see, Kirker-Head, C. A., Gerhart, T. N., Armstrong, R., Schelling, S. H. & Carmel, L. A. Healing bone using recombinant human bone morphogenetic protein 2 and copolymer. Clin. Orthop. Relat. Res., 205-17 (1998); Kokubo, S. et al. Bone regeneration by recombinant human bone morphogenetic protein-2 and a novel biodegradable carrier in a rabbit ulnar defect model. Biomaterials 24, 1643-51 (2003)). Since the commercially available BMP2 at present is very expensive, for example, the price of BMP2 available from Sigma for experimental research purposes reached US$ 500/10 μg, and few domestic patients can afford it. Moreover, several milligrams (mg) of BMP2s are equivalent to the total amount of BMP2 extracted from a bovine, and the safety issue becomes a concern due to the use of the large amount of BMP2.